Influenza A virus infection is an important cause of annual morbidity and mortality in the human population. Both inactivated and live attenuated vaccines are available but their effectiveness could be improved substantially to provide broader and longer lasting immunity. We have established a system for isolating and differentiating primary human nasal epithelial cell (hNECs) cultures with the goal of gaining a better understanding of the viral and cellular factors that are critical for virus replication. Our data demonstrate that live, attenuated influenza vaccine strains (LAIV) show a much greater degree of attenuation in hNEC cultures than they display in other cell culture systems, suggesting there are nasal epithelial cell specific factors contributing to the attenuated replication that have yetto be identified. This attenuation appears to be at the level of producing infectious virus particles because there appears to be little difference in secreted viral proteins but a 10,000 fold reduction in infectious virus production after LAIV infection of hNEC cultures. In Aim1 we will assess the innate immune factors that are differentially induced after infection of hNECs with wild type or LAIV strains of influenza to identify cellular factors that might be mediating this difference. In Aim 2 we will focus on identifying the antiviral signaling pathways and cytokines/chemokines that are differentially induced. In both Aims we will not only identify factors but use a variety of genetic and protein manipulations to demonstrate the relative contribution of each factor to LAIV attenuation. Finally, in Aim 3 we will characterize the defect in infectious virus production and determine what viral gene segments and mutations are contributing to the hNEC specific defect in LAIV infectious virus particle production. Our data wil allow us to identify novel cellular and viral factors that will provide targets for influenza virus therapeutics and that can be used to adjust the replication of LAIV in a manner that will improve its effectiveness as a vaccine. PUBLIC HEALTH RELEVANCE: Influenza is an important cause of disease in the human population. The goal of this proposal is to gain an understanding of how influenza virus and influenza vaccine strains differ in their ability to replicate in human nasal epithelial cells. Understanding the differences and similarities in how these two virus strains infect human nasal cells will provide insights into how to treat influenza as well as in how to engineer better vaccines.